Characterization of pigeon paramyxoviruses ( Newcastle disease virus ) isolated in South Africa from 2001 to 2006

C. ABOLNIK, G.H. GERDES, J. KITCHING, S. SWANEPOEL, M. ROMITO & S.P.R. BISSCHOP. 2008. Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006. Onderstepoort Journal of Veterinary Research, 75:147–152 Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

Large die-offs in doves and pigeons have occasionally been reported in various parts of South Africa since the 1980s after the first isolation of PPMV-1 from doves during an outbreak in September 1986 (Pienaar & Cilliers 1987).In the present study, the phylogenetic relationships between 21 South African PPMV-1 viruses isolated from doves, pigeons, chickens, a duck and an African ground hornbill (Bucorvus leadbeateri) were investigated, and the pathogenicity of the African ground hornbill isolate for chickens was determined.

Isolates
Virus isolation was performed at the Onderstepoort Veterinary Institute, Stellenbosch Provincial Veterinary Laboratory, the University of Pretoria's Poultry Reference Laboratory and Deltammune Laboratory by inoculation into the alantoic cavities of 9 to 10-day-old embryonated specific antibody-negative fowl eggs.Isolates are indicated in Table 1.

Reverse transcription-polymerase chain reaction and nucleotide sequencing
Viral RNA was extracted from alantoic fluid using TRIzol® reagent (Gibco, Invitrogen).The fusion (F) protein gene was targeted in a one-step RT-PCR using the oligonucleotide primer pair and thermal cycling parameters described elsewhere (Abolnik, Horner, Maharaj & Viljoen 2004).Cycle sequencing was performed using the ABI PRISM® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and the reverse primer from the RT-PCR to span the region containing the F 0 peptide cleavage site.Reactions were analysed with an ABI3130™ Genetic Analyser (Applied Biosystems).

Sequence analysis
Nucleotide and amino acid sequence analyses and alignment were carried out using Bioedit (Hall 1999) and ClustalW software (http://www.ebi.ac.uk/clustalw/index.html).
Phylogenies were reconstructed with MEGA 3.1 software (Kumar, Tamura & Nei 2004) using the neighbour-joining tree inference method with the Kimura 2-parameter substitution model and 1 000 bootstrap replicates to assign confidence levels to branches.

RESULTS AND DISCUSSION
Phylogenetic analysis of partial F protein genes indicated that the South African PPMV-1 isolates do not cluster together as a single geographical entity, but instead are split between the two lineages 4bi and 4bii (Fig. 1).One of the South Africa isolates, PIZA05N277, isolated from a pigeon in Pretoria (Gau teng Province) in May 2005 shared 99 % nucleotide sequence identity with the other international lineage 4bi viruses, but only 98-99 % and 94-95 % sequence identities with other South African lineage 4bi and lineages 4bii viruses, respectively.Therefore, we suspect that this strain was recently introduced into the country.The muscovy duck (Cairina moschata) (DKZA06N828) was a suspected organophosphate poisoning case and probably contracted PPMV-1 through contact with faeces from infected doves.CKZA06N606 was isolated from 4-week-old broilers and is only the second reported case of PPMV-1 infection of chickens in South Africa.The African ground hornbill isolate, BLZA06N779 shared 99.4 % sequence identities in the partial fusion protein gene with DOZA06N757 isolated from doves on the same farm during the outbreak.An MDT value of 89.4 h was obtained for BLZA06N779, which falls within the 90h time limit for mesogenic viruses.The ICPI value obtained was 0.33, which indicates that it is not velogenic but does not differentiate between mesogenic and lentogenic viruses.
The South African lineage 4bii strains formed a single clade and these viruses were mainly obtained from the Western Cape Province, apart from single isolates from Gauteng (-ZA06N690) and KwaZulu-Natal Provinces (ZA469/PPMV1/02).Nucleotide sequence homology within the South African clade varied from 96.7 % to 99.1 %.Relatively longer branch lengths suggest that the lineage 4bii strains may have been circulating in South Africa for an extended period.The sequences of 112 RRQKRF 117 and 112 RRKKRF 117 at F 0 for lineages 4bi and 4bii, respectively (Fig. 2), are in agreement with other reports (Collins, Strong & Alexander 1994 Although the bootstrap values supporting the phylogenetic relationships within lineages 4bi and 4bii are low, amino acid sequences (Fig. 2) support the phylogenetic relationships: lineage 4bi clade (c) is distinguished from clades (a) and (b) (Fig. 1) by T 3 , L 10 and R 27 substitutions, and lineage 4bii clade (d) differs from clade (e) by a P 36 →S substitution.
We have demonstrated that exotic strains of pigeon paramyxoviruses were introduced into South Africa on multiple occasions.Routes of introduction into South Africa are speculative, although they are most likely via the importation of infected pigeons for racing and ornamental purposes as reported in other countries (Aldous et al. 2004).A second case of PPMV-1 infection of chickens in South Africa is reported here, highlighting the importance of the threat of PPMV-1 to poultry, but PPMV-1 also potentially threatens biodiversity of wild birds, particularly rare indigenous dove and pigeon species.We report the first isolation of PPMV-1 from an African ground hornbill (Bucorvus leadbeateri), an increasingly rare species, which became acutely infected with PPMV-1 and died, although the virus did not display increased pathogenicity for chickens.It is likely that the ground hornbill became infected by scavenging off the dead doves in the area.Our findings suggest that PPMV-1 tests were performed according to standard procedures (OIE manual of standards: diagnostic tests and vaccines 2000).

FIG. 1
FIG.1Phylogenetic tree of a 374-bp region of the fusion protein gene of PPMV-1 viruses.South African strains are framed (bold type)